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Roger A. Sheldon
Delft University of Technology
&
CLEA Technologies
Green Bio-based Solutions for a Sustainable Economy
www.cleatechnologies.com 1
Green chemistry efficiently utilises
(preferably renewable) raw materials,
eliminates waste and avoids the use
of toxic and/or hazardous solvents
and reagents in the manufacture and
application of chemical products.
Green (Clean) Chemistry
Sheldon, Arends and Hanefeld , Green Chemistry
and Catalysis, Wiley, New York, 2007
Anastas & Warner, Green Chemistry : Theory
& Practice ,Oxford Univ. Press,New York,1998
2
3
Green (Clean) Chemistry
• Eliminating (minimizing) waste • Avoiding toxic / hazardous substances • Conserving raw materials
Definition of Waste ?
Meeting the needs of the present
generation without compromising
the needs of future generations to
meet their own needs
Sustainability
Brundtland Report, ‘Our Common Future’, 1987
5
The Bio-based Economy
Renewable Biomass
Biocatalysis
Chemocatalysis
Bio-based Products
Utilising crops and waste biomass which don’t compete with food
2nd generation:
Tonnage E Factor
Oil Refining 106-108 <0.1
Bulk Chemicals 104-106 <1 - 5
Fine chemical Industry 102-104 5 - >50
Pharmaceutical Industry 10-103 25 - >100
R.A.Sheldon, Chem & Ind, 1992, 903 ; 1997, 12
E Factor = kg waste/kg product
“Another aspect of process development mentioned by all pharmaceutical process chemists who spoke with C&EN is the need for determining an E Factor”. A. N. Thayer, C&EN, August 6, 2007, pp. 11-19
6
What about process water? Only counts if it needs to be treated?
Major Sources of Waste • Stoichiometric Reagents
- Acids & Bases
- Oxidants & reductants
• Solvent losses ( 85% of non-aqueous mass)
- Air emissions & aqueous effluent
• Multistep syntheses
The Solution :
Atom & step economic catalytic processes
in alternative reaction media
(the best solvent is no solvent)
7
The Environmental Impact EQ
EQ = E(kg waste) × Q
Q = Unfriendliness Multiplier
e.g. NaCl : Q = 1 ( arbitrary)
Cr salts : Q = 1000?
R.A.Sheldon, Chem & Ind, 1992, 903 ; 1997, 12
There are many shades of green!
Biocatalysis 9
Biocatalysis is Green & Sustainable • Enzymes are derived from renewable resources and are biodegradable (even edible sometimes) • Avoids use of (and product contamination by)
scarce precious metals • Mild conditions: ambient T & P in water
• High rates & highly specific : substrate, chemo-, regio-, and enantiospecific • Higher quality product • No special equipment needed
10
Reduced environmental footprint
Two Types of Biotransformations • Free enzymes - isolated (purified) - whole cells (not growing) - can be very high STY • Fermentations (growing microbial cells) - less expensive (no enzyme isolation needed) - often dilute solution / low STY - water footprint /energy intensive - byproducts from enzyme impurities
11
Lord, I fall upon my knees And pray that all my syntheses May not be inferior To those conducted by bacteria
J.B.S. Haldane
E factors of Fermentations
Product E factor Citric acid - excluding H2O (75% is CaSO4 ) 1.4 - including H2O 17 Bioethanol - excluding H2O and CO2 1.1 - including H2O and CO2 42a Recombinant Human Insulin - excluding H2O 6600b - including H2O 50.000
b 1692 kg urea, 1346kg HOAc, 968 kgs HCO2H, 713 kg H3PO4 ,
445 kg guanidine-HCl, 432 kg glucose, 430 kg NaCl, 424 kg MeCN
a 10000 ton/day lignocellulose feedstock produces 870 ton/day bioethanol
and generates 32x106 ltr/day of wastewater (eq. to 300.000 inhabitants)
12
Enzymatic VS Chemical Process for 6-APA
1. n-BuOH, -40 oC
2. H2O, 0 oC
Process Chemical Enzymatic
Reagents (kg/ kg 6-APA)
Me3SiCl (0.6) PCl5 (1.2) PhNMe2 (1.6)
n-BuOH (8.4 ltr), NH3 (0.2)
Pen acylase (1-2) NH3 (0.09)
Solvent (ltr/kg 6-APA)
CH2Cl2 (8.4) H2O (2)
pen acylase H 2O
1. Me3SiCl
2. PCl5 /
PhNMe 2/
CH2Cl2oo
37 C-40 C
6-APA
penicillin G
H2N
N
O
S
CO2H
Cl
N
N
O
S
CO2SiMe3
H
O
N
N
O
S
CO2H
13
Key improvements: enhanced enzyme production and immobilization
Reactions used for Drug Candidate Molecules
“A common synthetic sequence is the
conversion of an ester to an acid, activation of
the acid and conversion to the amide.”
Enzymatic amidation :
Lipases/esterases, Amidases, Proteases
“Three synthetic steps and multiple stoichiometric
components to accomplish a simple dehydration.”
14
Carey, Laffan, Thomson, Williams
Org. Biomol. Chem. 2006, 4, 2337
Enzymatic Amidation
de Zoete, Kock van Dalen, van Rantwijk and Sheldon, J.Mol.Cat.B, 2, 19 and 141 (1996)
15
van Pelt, Teeuwen, Janssen, Sheldon, Dunn, Howard, Kumar, Martínez, Wong, Green Chem. 13 (2011) 1791-1798
broadening the scope of enzymatic amidation
Protein Engineering 16
Production of Lipitor Intermediates
NC
OH O
OEt N C
O O
O R
O
Lipitor (Pfizer)
Sales in 2009: $14 bio
H N
O
N C O2 N a
O H O H
F
17
Enzymatic Synthesis of Lipitor Intermediate
KRED = keto reductase ; GDH = glucose dehydrogenase HHDH = halohydrin dehalogenase (non-natural nucleophile)
OEt Cl
O O
N A D P H N A D P
OEt Cl
OH O
OEt Cl
OH O
HHDH OEt
NC
OH O
KRED
g l u c o s e g l u c o n a t e
GDH
a q . N a C N , p H 7
(99.8% ee)
> 9 9 % e e
R.J.Fox, S.C.Davis,R.A.Sheldon, G.W.Huisman, et al Nature Biotechnology, 25 (2007) 338-344
2006
18
• high enantioselectivity
• mild (ambient) conditions
• no metal catalysts required
• no need for dedicated equipment
• low productivities
Directed Evolution for Improved Performance
19
Features of the Wild-Type Enzymes:
Productivities of all three enzymes improved by
directed evolution using gene shuffling technology
W.P.C.Stemmer,Nature,370,389-391,1994
Waste Quantity
( kg per kg HN)
% contribution to E
(excluding water)
% contribution to E
(including water)
ECAA losses (8%) 0.08 <2% <1%
Triethanolamine 0.04 <1% <1%
NaCl and Na2SO4 1.29 22% ca. 7%
Na-Gluconate 1.43 ca. 25% ca. 9%
BuOAc
(85%recycle)
0.46 ca. 8% ca. .3%
EtOAc
(85%recycle)
2.50 ca. 43% ca. 14%
Enzymes 0.023 <1% <1%
NADP 0.005 0.1% <0.1%
Water 12.25 - 67%
E Factor 5.8 (18)
E factor of the Codexis Three-Enzyme Process
R. A. Sheldon, G. Huisman et al, Green Chem. 2010, 12, 81-86
Presidential Green Chemistry Challenge Award 2006
20
Biocatalyst Engineering
21
The Challenge
Disadvantages of Enzymes
Low operational stability & shelf life
Cumbersome recovery & re-use
Product contamination
Allergic reactions to proteins
Non viable biocatalytic applications
Costs are too high
Not practical
The Solution: Immobilization
Cross-Linked Enzyme Aggregates (CLEAs)
Precipitation X-linking + Co-polymerization
• “Simple” & Broadly Applicable
• Cost-effective (no need for pure enzyme)
• Short Time-to-Market (low development costs)
• Scalable Protocols
e.g. (NH4)2SO4
or tert-butanol
Advantages of CLEAs
1. Improved properties
• Better storage and operational stability
• Hypoallergenic
• No leaching of enzyme in aqueous media
2. Cost-effective
• No need for pure enzyme (crude cell lysate sufficient)
• Easy recovery and recycle (easier DSP)
• High productivities (kg product/kg enzyme)
3. Broad scope & short time to market
0
20
40
60
80
100
120
0.0 5.0 10.0 15.0 20.0
Exposure to 21oC (days)
Re
sid
ual
Act
ivit
y (%
)
Free enzyme (%) Whole cell (%) Nitrile Hydratase CLEA (%)
4 months storage no decrease in activity!
Dramatic stability enhancement of a NHase
Dramatic Stability Enhancement of a NHase
25
Instability due to dissociation of multimeric enzyme
hindered by CLEA formation
van Pelt, Quignard, Kubac, Sorokin, van Rantwijk and Sheldon, Green Chem. 10 (2008) 395-400
Mitsubishi process: acrylonitrile to acrylamide > 200000 tpa
Cross-Linked enzyme Aggregates
AlcalaseCLEA commercial scale manufacture
- Free flowing powder
- Tunable particle size
- Low (no) allergenicity
- Excellent thermal stability 26
Nuijens et al, Advan. Synth. Catal. 2010, 352, 2399 – 2404.
Peptide synthesis with alcalase CLEA (DSM)
27
Cross-Linked enzyme Aggregates
Protease CLEAs as
Antifouling Agents in Paint
Cross-linked enzyme aggregates (CLEAs) of proteases were tested
in artificial seawater (ASW) both as it is and as a component of the
paint. It is found that all CLEAs have tolerance to xylene and have
great stability in dried paint.
The maximal increase in relative activity was found for CLEA B. licheniformis.
CLEA B. licheniformis have shown 900% activation during storage in ASW. In
the paint, non-modified subtilisin lost more that 90% of activity in 28 days.
S. Shipovskov, et al, J. Mater. Chem., 2010, 20, 7626–7629
28
29
The stone age didn’t end when there were no more stones left
Utilisation of Biomass for Sustainable Fuels & Chemicals
CM 0903 (UBIOCHEM)
The Plant Bottle
PET bottles
Crude oil
naphtha
ethylene
ethylene glycol
p-xylene
p-terephthalic acid
polyethylene terephthalate
Lignocellulosic biomass
cellulose
hemicellulose
C5 & C6
sugars
ethanol
ethylene
bio-ethylene glycol
isobutanol
fermentation
bio-p-xylene
APR
interm.
GEVO VIRENT
bio-formate
bio-p-xylene
Up to 30% plant-based
100% plant-based
same polymer advantages/disadvantages
An alternative biopolymer: Avantium process
C5 & C6 sugars
ethanol
ethylene
bio-ethylene glycol
ROH / chemocatalyst
PEF
Not the same polymer
Advantages /disadvantages: - Reduced carbon footprint - Better gas barrier properties - Recyclable - Not biodegradable
32
Take Home Message
Green chemistry, biocatalysis & valorization
of waste biomass:
Green Bio-based Solutions
for a Sustainable Economy
“A good company delivers excellent products and services. A great company does all of this and strives to make the world a better place”
William Ford Jr.
Thank you for your attention 33
and Sustainable